Papillomaviruses (PV) are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been recognized in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types of papillomavirus (HPV) that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott—Raven Pub., Philadelphia, N.Y.).
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into “low risk” and “high risk” types. The low risk types include HPV type 6, type 11 and type 13 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States there are approximately 5 million people with genital warts of which 90% is attributed to HPV-6 and HPV-11. About 90% of SIL is also caused by low risk types 6 and 11. The other 10% of SIL is caused by high risk HPVs.
The high risk types are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and 18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide (Fields, 1996, supra).
Treatments for genital warts include physical removal such as cryotherapy, CO2 laser, electrosurgery, or surgical excision. Cytotoxic agents may also be used such as trichloroacetic acid (TCA), podophyllin or podofilox. Immunomodulatory agents are also available such as Interferon or Imiquimod. These treatments are not completely effective in eliminating all viral particles and there is either a high cost incurred or uncomfortable side effects related thereto. In fact, there are currently no effective antiviral treatments for HPV infection. With all current therapies recurrent warts are common (Beutner & Ferenczy, 1997, Amer. J. Med., 102(5A):28-37).
The ineffectiveness of the current methods to treat HPV infections has demonstrated the need to identify new means to control or eliminate such infections. In recent years, efforts have been directed towards finding antiviral compounds, and especially compounds capable of interfering with viral replication (Hughes and Romanos, 1993, Nucleic Acids Res. 21:5817-5823; Clark et al., Antiviral Res., 1998, 37(2):97-106; Hajduk etal., 1997, J. Med. Chem., 49(20):3144-3150 and Cowsert et al., 1993, Antimicrob. Agents. Chemother., 37(2):171-177). To that end, it has therefore become important to study the genetics of HPVs in order to identify potential chemotherapeutic targets to contain and possibly eliminate any diseases caused by HPV infections.
The life cycle of PV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, cellular division continues as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation, the cellular machinery is maintained which allow viral gene expression to increase, with eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles (Fields, supra).
The coding strand for each of the papillomavirus contains approximately ten designated translational open reading frames (ORFs) that have been classified as either early ORFs or late ORFs. The E1 to E8 genes are expressed early in the viral replication cycle. The two late genes (L1 and L2) code for the major and minor capsid proteins respectively. The E1 and E2 gene products function in viral DNA replication, whereas E5, E6 and E7 modulate host cell proliferation. The L1 and L2 are involved in virion structure. The functions of E3, E4 and E8 gene products is uncertain at present.
Studies of HPV have shown that proteins E1 and E2 are the only viral proteins required for viral DNA replication in vitro (Kuo et al., 1994, J. Biol. Chem. 30: 24058-24065). This requirement is similar to that of bovine papillomavirus type 1 (BPV-1). Indeed, there is a high degree of similarity between E1 and E2 proteins and the ori-sequences of all papillomaviruses (PV) regardless of the viral species and type (Kuo et al., 1994, supra). Of note, E1 is the most highly conserved protein in PV and its enzymatic activity is presumed to be similar for all PV types (Jenkins, 1996, J. Gen. Virol. 77:1805-1809). It is therefore expected that all E1 gene products from different PV have similar structure and function. In addition PV E1 protein shows sequence and structural similarities to the simian virus 40 and polyomavirus large T protein (Clertant and Seif, 1984, Nature 311:276-279 and Mansley et al., 1997, J. Virology 71:7600-7608).
The E2 protein is a transcriptional activator that binds to E1 protein, these two proteins and the ori sequence form a ternary complex (Mohr et al., 1990, Science 250:1694-1699). It is believed that E2 enhances binding of E1 to the BPV origin of replication (Seo et al., 1993b, Proc. Natl. Acad. Sci., 90:2865-2869). In HPV, Lui et al. suggested that E2 stabilizes E1 binding to the ori (1995, J. Biol. Chem. 270(45):27283-27291 and McBride et al., 1991, J. Biol. Chem 266:18411-18414).
Evidence emanating from studies of BPV-1 have shown that E1 possesses ATPase and helicase activities that are required in the initiation of viral DNA replication (Seo et al., 1993a, Proc. Natl. Acad. Sci. USA 90:702-706; Yang et al., 1993, Proc. Natl. Acad. Sci. 90:5086-5090; and MacPherson et al., 1994, Virology 204:403-408).
The E1 protein from BPV is a phosphorylated nuclear protein having replication related functions. These include DNA and ATP binding, and, ATPase and helicase activities. Deletion mapping studies have identified the amino acids 121-311 as the region required for DNA binding. Mutations within this region obviate DNA binding by full length E1 protein (Leng et al, 1997, J. Virol. 71:848-852 and Thorner et al., 1993, Proc. Natl. Acad. Sci. USA 90:898-902). The second function, ATP binding and ATPase activities are essential for viral DNA replication. Point mutations within conserved regions in the ATP binding domain, inactivate the ability of E1 to bind or hydrolyze ATP with the concomitant loss of DNA replication (MacPherson et al., 1994, Virology 204:403-408; Raj and Stanley, 1995, J. Gen. Virol. 76:2949-2956 and Sun et al., 1990, J. Virol 64:5093-5105). The third activity possessed by the E1 protein, is the helicase activity or the unwinding of DNA ahead of the replication fork. Studies have predicted that helicase activity resides from the DNA binding domain, amino acid 121 through the ATPase/nucleotide binding region, approximately amino acid 530 (Sverdrup and Myers, Human Papillomaviruses, 1997, Published by Theoretical Biology and Biophysics).
When viral DNA replication proceeds in vitro, where E1 protein is present in excess, replication proceeds in the absence of E2. In vivo, in the presence of a vast amount of cellular DNA, replication requires the presence of both E1 and E2. E2, acts as a specificity factor in directing E1 to the origin of replication (Sedman and Stenlund, 1995, Embo. J. 14:6218-6228). The mechanism for initiating replication in vivo, is believed to involve the cooperative binding of E1 and E2 to the origin, whereby E1 and E2 form a complex. These interactions of DNA-protein and protein-protein occur at the origin of DNA replication (Sverdrup and Myers, supra).
Understanding the mechanisms of the E1 protein as a helicase, presumably capable of unwinding the DNA at the origin and ahead of the replication fork, is one of the advantages of this invention. Based on PV studies and SV40 DNA replication, a biphasic model for replication initiation for PV has been proposed (Sverdrup and Myers, supra). In a first step, E1 and E2 cooperatively bind to the origin of replication, thus ensuring binding specificity towards the origin of replication. In a second step, additional E1 monomers are recruited to the origin with the concomitant loss of E2. It is thought that the formation of the E1 homo-oligomeric complex at the origin is required for DNA replicating activity and the recruitment of the cellular replication machinery in initiating DNA synthesis (Sverdrup and Myers, supra).
Since there are as yet no effective therapeutic agents to prevent, control, decrease or eliminate PV infection, it has become important to study the life cycle of PV in greater detail and to specifically develop a better understanding of viral DNA replication. There is surprisingly little knowledge about the mechanism of E1 oligomerization in-vivo or in-vitro. The prior art is silent as to the location of the region along the E1 protein that is necessary for this protein-protein interaction, in the formation of the E1 oligomeric complex.
There thus remains a need to provide an understanding of the mechanism and the element/s involved in this oligomerization. Knowledge of this process provides a potentially new therapeutic target against PV.
It is therefore, one of the advantages of the present invention to identify an amino acid region in the E1 protein necessary for this apparent self-association.
Further, localization of this region by the Applicant provides a potential new therapeutic target in the treatment of PV infections. It is therefore a further advantage of the invention to provide a screening method for identifying agents capable of modulating this new target and a system to select at least one such agent capable of interfering with PV DNA replication.
The present invention refers to a number of documents, the content of which is herein incorporated by reference.